The present invention provides compositions which allow for the extended release and enhanced bioavailability of biologically-active polypeptides following parenteral delivery to an animal. More particularly, it concerns compositions comprising biologically-active somatotropin formulated for extended release, methods of preparing these compositions, and methods of using the same. These compositions comprise somatotropin, a bioavailability-enhancing constituent (BEC), and a substantially non-aqueous, hydrophobic excipient. The BEC may comprise (i) amino acids or amino acid derivatives, such as histidine-HCl; (ii) hydroxamate derivatives, such as histidine hydroxamate or suberohydroxamic acid; (iii) non-reducing carbohydrates, such as trehalose or trehalose octaacetate; (iv) oxo-acid salts, such as a mixture of monobasic and dibasic sodium phosphate; or (v) a mixture of two or more compounds from within the foregoing classes (i) (iv).

SUMMARY OF THE INVENTION

The present invention provides for a composition of matter, which provides both for higher serum levels of somatotropin ("ST") and for a prolonged maintenance of those higher serum ST levels, compared to known formulations containing the same dose of somatotropin. Furthermore, these formulations have been demonstrated to also have superior efficacy for inducing weight gain and milk production when compared with currently available formulations containing the same amount of somatotropin. The ST formulations of the present invention have been demonstrated to be particularly effective for sustaining elevated milk production, compared to previously available formulations.

The formulations of the present invention provide for compositions of matter which comprise somatotropin and a bioavailability-enhancing constituent (BEC, where "bioavailability" is the net result of the release, absorption, elimination, degradation and other physiological processes for a substance). The bioavailability-enhancing constituent can be comprised of one or more of the following: (a) an amino acid, amino acid derivative such as histidine-HCl, or an amino acid polymer, such as polyhistidine; (b) an hydroxamate, such as suberohydrxamic acid, or hydroxamate derivative, such as histidine hydroxamate; (c) a non-reducing carbohydrate, such as a polyol or polyol ester; (d) the salt of an oxo-acid, such as monobasic sodium phosphate or dibasic sodium phosphate or a mixture of monobasic and dibasic sodium phosphate; or (e) imidazole or imidazole-HCl.

In various embodiments of the invention the ST and the BEC are suspended in a substantially non-aqueous hydrophobic carrier which yields a formulation which is fluidly injectable at the body temperature of the animal to be injected (typically 37 39.degree. C.), and preferably injectable at 25.degree. C. The compositions of the instant invention may be of any viscosity which is compatible with the present invention. In a preferred embodiment the viscosity of the composition is between about 500 and about 10,000 centipoise at 141.sup.-1 s. An exemplary carrier considered for use in the present invention is a mixture of 95% sesame oil and 5% aluminum monostearate

According to the present invention the ST used in the formulations may be from any origin which is suitable for use with the invention, including, but not limited to, native and/or recombinant bovine, porcine, equine, or human somatotropin. The ST used may be present in an essentially pure form or may be combined with another substance (e.g., the ST used may be in the form of a zinc salt or zinc complex of ST).

In an alternative embodiment, where the BEC is an amino acid, amino acid derivative, hydroxamate or hydroxamate derivative, the BEC comprises from about 0.5% to about 30%; preferably from about 1% to about 20%; and even more desirably from about 2% to about 10% of the composition.

According to another embodiment of the present invention, when the BEC used is a non-reducing carbohydrate it comprises from about 1% to about 50%, of the composition, preferably from 5% to 20%, and even more preferably from 10% to 15% of the composition.

In yet another embodiment of the present invention, when the BEC is the salt of an oxo-acid, the BEC comprises from about 0.5% to about 30% of the composition, preferably from about 1% to about 20%, and even more preferably from about 2% to about 10% of the composition.

The present invention also provides a method of preparing the described compositions of matter. According to such an embodiment of the present invention the ST is provided as a lyophilized dry solid (for an example of preparing the lyophilized ST see U.S. Pat. No. 5,013,713, which is incorporated herein by reference), the BEC is likewise provided as a dry solid. The method for preparing these compositions of matter comprises mixing the dry, solid BEC with the hydrophobic carrier, to produce a first suspension and then mixing the lyophilized, dry, solid ST with the first suspension to produce a second suspension.

Another embodiment of the present invention provides a method for inducing improved weight gain or elevated milk production in a mammal. This method comprises injecting an ST formulation of the present invention into the target mammal.

Yet another embodiment of the present invention provides a method for sustaining the elevated milk production response in a lactating mammal. This method comprises injecting the target mammal with a biocompatible ST formulation according to the present invention, wherein the ST present in the formulation is active in the target mammal.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides an injectable (or otherwise parenterally administerable) enhanced bioavailability formulation comprising: (i) a dry solid native or recombinant protein growth hormone (GH, synonymous with somatotropin, herein sometimes abbreviated as ST), and (ii) a dry, solid bioavailability-enhancing constituent (BEC). Both the ST and BEC are suspended in an excipient comprised of a substantially non-aqueous oil, fat, or other hydrophobic substance that is at least partially liquid to creamy and/or biodegradable in an animal at the animal's body temperature and which is biocompatible with the animal.

Somatotropins from man and from the common domestic animals are proteins of approximately 191 amino acids, which are synthesized and secreted by the anterior lobe of the pituitary gland. Full length human ST (hST) consists of 191 amino acids. ST is a key hormone involved in the coordination of somatic growth through the regulation of the metabolic processing of proteins, carbohydrates, and lipids. A major known effect of ST is the promotion of growth in organ systems which include, but are not limited to, the skeleton, connective tissue, muscles, and viscera such as the liver, intestine, and kidneys.

Growth hormones suitable for use in the present invention include, but are not limited to, somatotropins from human, bovine, equine, ovine, porcine, caprine, and avian sources. Preferably the ST is human, equine, bovine, or porcine ST. Even more preferably the ST is bovine or human ST (bST).

Somatotropin for use in the present invention can be obtained by extraction and subsequent concentration from the pituitary glands of various animals. Alternatively, ST may be produced using recombinant DNA techniques which are well understood and commonly used by those skilled in the art.

Somatotropins prepared using recombinant DNA techniques suitable for use according to the present invention may be produced by genetically transformed microorganism such as E. coli, or other bacteria, or by yeasts. Recombinant ST may also be produced by tissue culture or by a transgenic multicellular organism.

Somatotropins produced using molecular biological techniques may have an amino acid sequence identical to naturally occurring somatotropin. Alternatively, the somatotropin used may be an ST analog comprising one or more variations in amino acid sequence with respect to the native hormone. These amino acid variations may provide enhanced biological activity or some other biological or logistical advantages.

ST is usually synthesized in its native organism as an inactive precursor molecule which is processed to the mature, active form of the hormone via cleavage of an N-terminal signal peptide (26 amino acids in humans, 27 amino acids in cows). In order to express bioactive ST protein using recombinant DNA technology it may be advantageous to add, change, or delete one or more amino acids from the polypeptide. Such modifications, which do not overly diminish the activity of the ST, or make it biologically incompatible with the animal to be treated, are useful in the present invention. For example it may be useful to produce an ST polypeptide which contains a methionine residue at its N-terminus (N-terminal to the phenylalanine residue which is typically the first amino acid in the mature, active form the native hormone), resulting from microbial translation of the AUG start codon in a recombinant gene for the polypeptide (this form of ST is known as N-methionyl-ST). Another derivative envisioned for use in the present invention is N-alaninyl-ST, which is similar to N-methionyl-ST except it begins with an N-terminal alanine residue (See Krivi U.S. Pat. No. 5,399,489, which is incorporated herein by reference) rather than an N-terminal methionine.

According to the various embodiments of the current invention the ST can administered in a chemically uncombined form. Other embodiments are advantageously carried out using ST in a form which has substantially lower solubility in aqueous environments or animal body fluids than the uncombined ST (e.g. chemically or otherwise combined with another substance). For example, the ST can be predominantly or partially chemically associated with a biocompatible metal, or an ester, amide or other moiety or moieties which aid in providing the desired bioactivity and which do not induce intolerable side effects in the animal treated with the composition. When associated with such a metal, the metal can be present as the metal per se (e.g. as a metal salt of or in a complex with the ST) or in the form of a salt or complex of the metal with one or more other anions.

Although monovalent metals (e.g. sodium or potassium) can be used advantageously in some compositions of this invention, polyvalent metals are preferred. Examples of such polyvalent metals include zinc, iron, calcium, bismuth, barium, magnesium, manganese, aluminum, copper, cobalt, nickel, cadmium and the like. In certain highly preferred embodiments, such metal-associated ST molecules are reaction products of such metals, e.g. in ionic form, with dissolved ST. The ratio of metal to ST may vary depending on the number of active sites of the ST that associate with such metal during the formation process (e.g., it may vary as a function of pH). For instance, metal may be associated with some or all negatively-charged amino acid (e.g. aspartic or glutamic) residues in the ST, or with its carboxy terminus. Some or all of the metal may be associated by any physical or chemical means including, but not limited to, as salt or complex with the ST, occluded within folds, crystals or amorphous shapes of the ST, or associated as a cation bridge between at least two ST molecules.

When the metal is polyvalent, its valence may be only partly chemically associated with the ST polypeptide in some cases, e.g. because of steric hindrance. In such cases, the remaining valence of the metal may be chemically associated with other anions. In many desirable embodiments, the metal is not chemically associated in substantial proportion with other anions that form salts or complexes having low water solubility with said metal. When the metal is partly chemically associated with other anions, such other anions (organic or inorganic) are often desirably selected from those that form water-soluble salts or complexes with that metal, e.g. Br.sup.-, Cl.sup.-, I.sup.-, SO.sub.4.sup.2-, or CH.sub.3COO.sup.- when the metal is zinc. Monovalent anions, e.g. Cl.sup.-, are generally most preferred.

A preferred embodiment of this invention includes somatotropins associated with zinc (ZnST). In some instances, these may contain up to about 5% zinc or more, based on the weight of the somatotropin. To minimize the chance of undesirable injection site responses in the animals, however, it may be desirable for them to contain no more than about 2%, and in some instances no more than about 1% zinc. In preferred embodiments these ZnST molecules contain at least about 0.3% (usually at least about 0.5%) zinc, although lower percentages of zinc may be suitable in some cases.

Examples of other ST salts and complexes useful in this invention include: (i) acid addition salts formed with inorganic acids, e.g., hydrochloric, hydrobromic, sulfuiric, phosphoric or nitric; or organic acids, e.g., acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, ascorbic, benzoic, tannic, pamoic, alginic, polyglutamic, naphthalenesulfonic, naphthalene-disulfonic or polygalacturonic; (ii) salts and complexes with polyvalent organic cations, e.g. N'-dibenzylethylenediamine or ethylenediamine; and (iii) combinations of two or more of the aforementioned types of salts or complexes, e.g. zinc tannate.

Especially preferred are salts and complexes of zinc, iron, calcium, magnesium, manganese, sodium, potassium and mixtures thereof. Even more preferred, are salts or complexes of zinc, sodium or potassium, with ZnST being most preferred.

In a preferred embodiment of the present invention the bioavailability-enhancing constituent (BEC) comprises one or more substances selected from the following: (a) an amino acid or amino acid derivative, (b) an hydroxamate or hydroxamate derivative, (c) a non-reducing carbohydrate, (d) an oxo-acid salt, or (e) imidazole or imidazole-HCl.

Amino acids and amino acid derivatives which are compatible with and preferred as bioavailability-enhancing constituents for use with the instant invention include, but are not limited to the following: histidine, histidine salts, such as histidine-HCl, histidine derivatives, including polyhistidine and histidine hydroxamate, arginine, lysine, tryptophan, methionine, arginine, glutamic acid, aspartic acid, glycine. The use of histidine-HCl is particularly preferred.

Amino acids or amino acid derivatives can be added in an amount ranging from 0.5 to 30% (w/w) in the formulation. The more desirable range of amino acid or derivative is 1 20% (w/w) and the most preferred range is 2 10% (w/w). In formulations comprising amino acids or amino acid derivatives ST can be added in an amount ranging from 10 to 50% (w/w) in the formulation. The more desirable range for ST is 20 46% and the most preferred range is 32 42% (w/w). Thus, the most preferred non-aqueous injectable formulation preparation, having amino acids or amino acid derivatives, comprises amino acid or derivative in an amount of 0.05 to 0.3 mg (2 10% by weight) per mg of ST (32 42% by weight).

In another embodiment of the present invention the bioavailability-enhancing constituent is a hydroxamate or hydroxamate derivative. Preferred hydroxamates for use as bioavailability-enhancing constituents in the present invention are those which bind metals, particularly, zinc, these include: suberohydroxamic acid, salicly hydroxamic acid, bufexamac acid, and caprylohydroxamic acid.

Hydroxamates or hydroxamate derivatives can be added in an amount ranging from 0.5 to 30% (w/w) in the formulation. The more desirable range of hydroxamates or hydroxamate derivatives is 1 20% (w/w) and the most preferred range is 2 10% (w/w). In formulations comprising hydroxamates or hydroxamate derivatives, ST can be added in an amount ranging from 10 to 50% (w/w) in the formulation. The more desirable range for ST is 20 46% and the most preferred range is 30 42% (w/w). Thus, the most preferred non-aqueous injectable formulation preparation having hydroxamates or hydroxamate derivatives, comprises hydroxamate or hydroxamate derivative in an amount of 0.05 to 0.3 mg (2 10% by weight) per mg of ST (32 42% by weight).

Another embodiment of the present invention provides for a composition comprising a non-reducing carbohydrate as the bioavailability-enhancing constituent. Non-reducing carbohydrates which are useful in the compositions of the present invention include, but are not limited to: polyols and their ester derivatives. Polyols which are preferred for use in the present invention include, but are not limited to, trehalose, sucrose, mannitol and sorbitol. Trehalose (in the form of trehalose dihydrate) is particularly preferred. Preferred polyol esters include: acetate, octaacetate, and octasulfate polyol esters. Particularly preferred polyol esters include trehalose octaacetate, sucrose octaacetate, and cellobiose octaacetate.

The non-reducing carbohydrates can be added from 1 to 50% (w/w) in the formulation. The more desirable range of non-reducing carbohydrates is 5 20% (w/w) and the preferred range of is 10 15% (w/w). In formulations comprising non-reducing carbohydrates, ST can be added in an amount ranging from 10 to 50% (w/w) in the formulation. The more desirable range for ST is 20 46% (w/w) and the most preferred range is 30 40% (w/w).

In another embodiment of the present invention the bioavailability-enhancing agent is an oxo-acid salt (where an oxo-acid is an acid in which the acidic hydrogen atoms are bound to oxygen atoms). Preferred bioavailability-enhancing agents of this embodiment include: (i) monobasic sodium phosphate, NaH.sub.2PO.sub.4, dibasic sodium phosphate, Na.sub.2HPO.sub.4, or a mixture of monobasic and dibasic; (ii) monobasic potassium phosphate, KH.sub.2PO.sub.4, dibasic potassium phosphate, K.sub.2HPO.sub.4, or a mixture of monobasic and dibasic; (iii) monobasic calcium phosphate, Ca(H.sub.2PO.sub.4).sub.2, dibasic calcium phosphate, CaHPO.sub.4, or a mixture of monobasic and dibasic; (iv) various other phosphate, sulfate or nitrate salts such as Ca.sub.3(PO.sub.4).sub.2, Na.sub.2SO.sub.4, and NaNO.sub.3, or mixtures thereof. In a preferred embodiment the bioavailability-enhancing agent is a mixture of monobasic- and dibasic sodium phosphate, in a mixture containing more monobasic than dibasic. It is important to note that as used in this embodiment the phosphate does not act as a buffering component and does not effect the pH of the composition. The pH is determined prior to adding the phosphate and is not significantly altered by the addition of the phosphate.

The oxo-acid salt can be added from 0.5 to 30% in the formulation. The more desirable range of oxo-acid salt is 1 20% and the preferred range of oxo-acid salt is 2 10%. ST can be added from 10 to 50% in the formulation. For ST, the more desirable range is 20 40%, and the preferred range is 32 42%.

The non-aqueous carrier (excipient) can be any substance that is biocompatible and liquid or soft enough at the animal's body temperature to release the somatotropin into the animal's bloodstream at a desired rate. The carrier is usually hydrophobic and commonly organic, e.g., an oil or fat of vegetable, animal, mineral or synthetic origin or derivation. Preferably, but not necessarily, the carrier includes at least one chemical moiety of the kind that typifies "fatty" compounds, e.g., fatty acids, alcohols, esters, etc., i.e., a hydrocarbon chain, an ester linkage, or both. "Fatty" acids in this context include acetic, propionic and butyric acids through straight- or branched-chain organic acids containing up to 30 or more carbon atoms. Preferably, the carrier is immiscible in water and/or soluble in the substances commonly known as fat solvents. The carrier can correspond to a reaction product of such a "fatty" compound or compounds with a hydroxy compound, e.g., a mono-hydric, di-hydric, trihydric or other polyhydric alcohol, e.g., glycerol, propanediol, lauryl alcohol, polyethylene or -propylene glycol, etc. These compounds include the fat-soluble vitamins, e.g., tocopherols and their esters, e.g., acetates sometimes produced to stabilize tocopherols. Sometimes, for economic reasons, the carrier may preferably comprise a natural, unmodified vegetable oil such as sesame oil, soybean oil, peanut oil, palm oil, or an unmodified fat. Alternatively the vegetable oil or fat may be modified by hydrogenation or other chemical means which is compatible with the present invention. The appropriate use of hydrophobic substances prepared by synthetic means is also envisioned.

Compositions of this invention may also comprise, in addition to the biocompatible oil, an "antihydration agent" which term as used herein means a substance that retards hydration of a somatotropin and/or the biocompatible oil or fat and thereby further decreases and/or stabilizes the rate of release of the ST from that composition following administration to an animal. A great variety of non-toxic antihydration agents are known. By way of example there are "gelling" agents which, when dispersed, and in some cases heated to dissolve them in the oil, give the body of oil greater visco-elasticity (and therefore greater structural stability) and thereby slow down penetration of the oil by body fluids.

The exact mechanism of these agents in the present invention is not fully understood. Thus it has been observed that certain known "gelling" agents provide the desired antihydration effect even when the oil containing such an agent has not been heated to enhance their gelling effect, or when the gel formation, once formed, has been substantially eliminated (e.g. by shear forces). Also, various antihydration agents that do not have substantial ability to gel the oil are suitable for use in this invention (magnesium stearate is one example).

Exemplary antihydration agents include various polyvalent metal salts or complexes of organic acids, for instance fatty acids having from about 8 (preferably at least about 10) to about 22 (preferably up to about 20) carbon atoms, e.g. aluminum, zinc, magnesium or calcium salts of lauric acid, palmitic acid, stearic acid and the like. Such salts may be mono-, di- or tri-substituted, depending on the valence of the metal and the degree of oxidation of the metal by the acid. Particularly useful are the aluminum salts of such fatty acids. Aluminum monostearate and distearate are particularly preferred anti-hydration agents. Others that are useful include aluminum tristearate, calcium mono- and distearate, magnesium mono- and distearate and the corresponding palmitates, laurates and the like. In many embodiments, the concentration of such an antihydration agent, based on the weight of the oil plus that agent, will be advantageously between about 1% and about 10% (most typically between about 2% and about 5%), although other concentrations may be suitable in some cases.

In one embodiment of the invention the non-aqueous carrier is present at about 40% to about 90% by weight, preferably the hydrophobic carrier is present at about 40% to about 70% by weight. In one preferred embodiment the carrier is selected such that the composition is a creamy suspension at 25.degree. C.; e.g., approximately 95% sesame oil gelled with 5% aluminum monostearate (AIMS).

Dry, solid somatotropin and dry, solid bioavailability-enhancing constituent can be prepared by lyophilization of appropriate solutions containing, respectively the somatotropin or the BEC. Alternatively, ST or the BEC can be prepared by any other means which provides them in the proper form for use in the present invention.

The compositions of matter described for the instant invention may be prepared by any means or procedure that provides for a composition which delivers the desired enhanced bioavailability of ST at the required levels. In a preferred embodiment of the invention a certain amount of the substantially dry, solid, BEC that is effective to further increase the bioavailability of bioactive ST is mixed with the non-aqueous excipient. Next, lyophilized ST is added and the mixture is milled for an appropriate period of time in order to achieve the desired particle size and viscosity of the composition (i.e., a particle size should be small enough to provide a viscosity which allows the formulation to be useful according to the current invention). A discussion of how to achieve the desired particle size may be found in U.S. Pat. No. 5,013,713 to James Mitchell, which is herein incorporated by reference for this purpose.

While the above method is preferred, the order of adding the BEC and the ST is flexible. Furthermore, ST and some BECs can be co-lyophilized first and then this co-lyophilized product can be mixed with the non-aqueous excipient and processed as described above.

The parenteral administration of the formulations, described herein, to an animal (e.g., a mammal such as a bovine) have shown that the compositions exhibit surprisingly improved bioavailability performance characteristics for ST when compared to previously available ST formulations.

In particular, they provide a relatively more rapid, yet controlled, release during the first several days after administration without the early release becoming an exaggerated "burst" to the extent that durability of the release is unduly impaired. Typically, in terms its practical effects, the most meaningful measure of the ST release over a given period of time is calculated as the area under the curve ("AUC") illustrating the amount of ST made available by the release (e.g., the serum level of ST in the animal) and/or a physiological consequence of the release (e.g., milk produced or weight gained by an animal in which the ST has been released). Use of the invention has been found to provide a surprisingly greater AUC for measured serum concentrations, this finding is particularly true for periods of time of 14 days or more.

Even more surprising is that when the AUC represents the desirable physiological effects of ST release (e.g., the amount of milk produced daily by bovine or other lactating animals) for animals treated with the ST formulations of the present invention, the AUC is not only surprisingly greater than those of known ST/oil formulations, but also surprisingly greater than would be expected, given the magnitude of the serum ST levels present in the lactating animal. Put another way, this means that the formulations of the present invention not only provide for an enhanced release of ST, but also increase the efficacy of the released amount of ST formulation. Consequently, despite declining serum ST levels, increased milk production is sustained at a level which is surprisingly high.

Examples of the enhanced physiological effects produced by compositions of the present invention are described in examples 3 5 below. These examples describe the unexpected results of sustained elevated milk production response in lactating animals treated with the ST compositions of the instant invention. In these examples serum ST levels and milk production of animals injected with the compositions of the present invention are compared with the serum ST levels and milk production of animals, that were either untreated, or were injected with the same dose of the same somatotropin in a composition which comprised essentially the same carrier, but which lacked the bioavailability-enhancing constituent. These examples demonstrate that the animals injected with the ST compositions of the present invention have an unexpectedly high and sustained level of milk production.
 

Claim 1 of 18 Claims

1. A method for administering somatotropin to a mammal that comprises injecting into a mammal a composition of matter comprising: samatotropin and a bioavailability enhancing constituent; wherein the bioavailability enhancing constituent comprises the following: a) one or more non-reducing carbohydrates, and/or b) one or more oxo-acid salts; wherein the somatotropin and the bioavailability enhancing constituent are suspended in a substantially non-aqueous hydrophobic carrier; wherein the somatotropin is present from about 10% to about 50% by weight of the composition; wherein the bioavailability enhancing constituent is present from about 1% to about 20% by weight of the composition; and wherein the composition is fluidly injectable at 25.degree. C.

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